Mass spectrometric measurement of microbial resistances

ABSTRACT

Microorganisms, particularly bacteria, are identified and characterized on the basis of a mass spectrometric measurement of their protein profiles with ionization by matrix-assisted laser desorption. In order to measure the microbial resistance to antibiotics, the protein profiles of microorganisms are measured after cultivation for a short time duration in nutrient media containing the antibiotics.

BACKGROUND

The invention relates to the identification and characterization ofmicroorganisms, particularly of bacteria, on the basis of a massspectrometric measurement of their protein profiles with ionization bymatrix-assisted laser desorption. Many types of microorganism (alsotermed microbes below), especially bacteria and unicellular fungi, canbe very easily identified mass spectrometrically using a recentlydiscovered method in which small quantities of microbes from a colonycultivated in the usual way on a nutrient medium are transferred to amass spectrometric sample support plate, where they are measureddirectly with a mass spectrometer. The mass spectrum particularlyrepresents the proteins of different masses if they are present insufficient concentration in the microbes. Spectral libraries withhundreds or thousands of microbe spectra are then used to establish theidentity of the microbes from this microbial protein profile.

The nutrient medium is usually in a moist gelatin in a Petri dish, thusmaking it possible to cultivate strains, each of which is pure, inseparate microbe colonies simply and in the completely normal way. Aquantity of microbes is transferred with a small spatula from a selectedcolony onto the mass spectrometric sample support, where the microbesare sprinkled with a solution of a conventional matrix substance forionization by matrix-assisted laser desorption (MALDI). The organicsolvent penetrates into the microbial cells and destroys them. Thesample is then dried by evaporating the solvent and hence the dissolvedmatrix material crystallizes. Soluble proteins and peptides and, to alesser extent, other substances of the cell are thus embedded into thematrix crystals.

The matrix crystals with the embedded analyte molecules are thenbombarded with flashes of laser light in a mass spectrometer, thuscreating ions of the analyte molecules which can then be measuredseparately according to the mass of the ions in the mass spectrometer.It is preferable to use time-of-flight mass spectrometers for thispurpose. The mass spectrum is the profile of the mass values of thesepeptide ions, protein ions and other analyte ions. This profile is verycharacteristic of the types of microbe in question because each type ofmicrobe produces its own, genetically predetermined proteins, eachhaving characteristic masses. The protein profiles are characteristic ofthe microbes in the same way that fingerprints are characteristic ofhumans. Many laboratories, including central, state-controlledinstitutions for disease monitoring and prevention, are working onreliable and legally applicable (so-called “validated”) massspectrometric libraries of protein profiles of microbes.

This simple method can even be used to distinguish between closelyrelated sub-strains of microbes since the proteins of the microbes aregenetically predefined and vary clearly in the sub-strains. Slightchanges in the genetic blueprint create proteins with a necessarilydifferent structure, whose masses are different to those of proteinswhose structure has not been genetically modified; they thus have adifferent protein profile. New taxonomic classifications of microbes caneven be performed in this way.

The masses of the proteins of completely identical types of microbe arenaturally always the same and thus strictly reproducible; theintensities of the protein signals, on the other hand, can only bereproduced approximately. The use of different nutrient media for thecultivation affects the metabolism of the microbes and hence affects thequantities of the different proteins which are produced and theirintensity in the protein profile. The effect is not great, however. Theintensity fluctuations do not interfere with the identification if thecomputer programs are adapted accordingly. Likewise, the degree ofmaturity of the colonies only affects the intensities of the proteinsignals with respect to each other in the mass spectra, but here, aswell, the effect is only slight. Mass spectra which have differentcharacteristics for the same type of microbe only really exist in thecase of spore-forming organisms: the spores have protein profiles whichare different to those of normal cells.

The computer programs for searching libraries by comparing spectra takeaccount of intensity fluctuations; the intensities play only a minorrole here. Current analyses have shown that the identification of themicrobes with these programs appears to be very reliable. The programsoperate only via the similarity of the mass spectra, withoutindividually identifying the proteins involved; the masses arerigorously incorporated into the search for similarities, theintensities much less rigorously. In particular, it is even possible forseveral proteins to be missing from the mass spectra (very lowintensity) without this interfering with the similarity determination:It suffices for the identification that the mass values for the majorityof proteins match. The library spectra are also able to storeinformation as to which protein signals definitely have to be present,for example by storing threshold values for the intensities.

The above, briefly described method of using a small spatula to spreadsome microbes from a colony onto a reserved spot of a mass spectrometricsample support, which is then sprinkled with a matrix solution, is thesimplest and, as yet, fastest type of sample preparation. The method canalso be automated with the help of image recognizing pipetting robotsfor use in routine laboratories. After cultivating a colony which isonly just visible, it takes only one or two hours until theidentification is complete even if hundreds of samples are analyzed atthe same time. Mass spectrometric sample supports each holding 384samples are commercially available; scanning these mass spectra takesaround one to two hours. If the task is urgent, individual microbesamples can be identified in a few minutes.

Other methods of sample preparation such as extracting the proteinsafter ultrasonic destruction (sonication) of the microbes, orcentrifugal extraction have also been investigated. These methodsprovide spectra which are without exception surprisingly similar.

Nowadays, the mass spectra of the microbe proteins are scanned in lineartime-of-flight mass spectrometers because these have a particularly highdetection sensitivity, although the mass resolution of the spectra andthe accuracy of the masses are considerably better from time-of-flightmass spectrometers with reflectors. In reflector mode, only around atwentieth of the ion signals appear, however, and the detectionsensitivity is one to two orders of magnitude worse. The highsensitivity of the linear mode of a time-of-flight mass spectrometer isdue to the fact that not only the stable ions are detected but also thefragment ions from so-called “metastable” decays of the ions. Even theneutral particles which are created en route from ion decays aremeasured. All these fragment ions and neutral particles, which haveoriginated from one species of parent ion, have the same speed as theparent ions and thus arrive at the ion detector at the same time. Thearrival time is a measure of the mass of the originally undecayed ions.

The increased detection sensitivity is so crucial for many applicationsthat many of the disadvantages of the linear mode of operation of thetime-of-flight mass spectrometer are tolerated. The energy of thedesorbing and ionizing laser is increased for these applications,something which increases the ion yield but also increases theirinstability, although this does not matter here.

Acquiring mass spectra with time-of-flight mass spectrometers generallyrequires that a very high number of individual spectra are measured inrapid succession, said individual spectra usually being added togethermeasurement point by measurement point to form a sum spectrum. The ionsfor each individual spectrum are generated by one laser bombardment foreach spectrum. The sum spectra have to be generated in this way becauseof the low dynamic range of measurement in the individual spectrum. Aminimum of approx. 50, in some cases even 1,000 or more individualspectra are measured here; in general, a sum spectrum consists ofseveral hundred individual spectra, which modern mass spectrometersmeasure and add together in a few seconds. The total duration of a sumspectrum acquisition depends on the number of individual spectra and thebombardment frequency of the laser used. Lasers with 20 to 200 hertz arenow used for this purpose; it takes around two to 20 seconds to acquirea good sum spectrum.

In the above-described fields of application, mass spectra are measuredwhich reach into high mass ranges of 20,000 Daltons, for example. Aspreviously mentioned, the low mass resolution means that in most partsof the mass spectrum it is no longer possible to resolve the isotopegroups, which consist of ion signals which each differ by one Dalton. Itis thus only the envelopes of the isotope groups which are measured.Mass spectrometric measuring methods have also become known whichprovide a higher resolution and a higher mass accuracy; it is not yetknown, however, if they achieve comparable sensitivities.

This method of quickly and simply identifying microbes can be used inmany areas, for example for monitoring drinking water or quality controlin food production. In the case of food production, it is the type ofmicroorganisms present which determines if the food is safe to consume.Suffice it here to mention harmful staphylococci, streptococci andsalmonellae, which have to be found by continuous controls. On the otherhand, beer, wine, cheese and yoghurt cannot be produced without thebeneficial deployment of billions of microbes. The crucial thing here isthat the strains are pure.

Strict monitoring is also necessary in the medical field. The main thingis to keep infective pathogens away from hospitals. Constant monitoringand identification of the ubiquitous microbes is a mandatory legalrequirement for operating rooms, for example.

The identification of microbes is particularly important with infectiousdiseases. It is important that the type of pathogen can be identifiedvery quickly in order that the correct medical care can be providedimmediately. Mass spectrometric identification has also provensuccessful here although it has not yet become established.

In the medical field, there is not only the problem of fastidentification, but also the problem of identifying resistances to thecommonly used antibiotics. It is not possible to fight the diseasequickly if the resistances are unknown. What is therefore required isnot only fast identification but also fast determination andcharacterization of the resistances of microorganisms.

Until now, the determination of the resistances has been predominantlybased on cultivation experiments of the microorganisms in differentnutrient media containing bactericides or other antibiotics. Theseexperiments are protracted and labor-intensive. They take at least 24hours, usually even two days. Experiments are currently being carriedout to identify the resistances using analyses of DNA sequences in theplasmids of the bacteria. The resistances are coded in the plasmids.This type of analytical method is very promising, but has not gainedacceptance as yet.

The problem of microorganisms being resistant to antibiotics such asbactericides or fungicides is becoming more and more critical as timegoes on. On the one hand, the speed with which microorganisms formresistance to different types of antibiotics is increasing; on the otherhand, fewer and fewer new antibiotics with a medical application arebeing developed. It is now known that the resistances are not formed bynew types of mutation and their selection, but by the interchange ofplasmids between microorganisms, even between different types ofmicroorganism. The microorganisms infect each other with theresistances.

There are many reasons for the rapid increase in resistances:irresponsible prescribing of antibiotics even when they are notnecessary; courses of treatment with bactericides which are rashlybroken off before the infective agents have been completely eradicated;irresponsible use in agriculture and livestock farming, often as apurely preventative measure. All these types of behavior help theselection of resistant types of microbes over the non-resistant types.

On the other hand, fewer and fewer new antibiotics are being developed.Since many new antibiotics have to be taken off the market after only ashort time because they became ineffective, it is becoming less and lessviable for the pharmaceutical companies to invest large amounts of moneyin developing antibiotics when this is becoming more and more difficult.According to the WHO, only three new antibiotics have come onto themarket since 1990, whereas there were ten between 1940 and 1950 and fivebetween 1971 and 1980.

Since the discovery of antibiotics by Alexander Fleming in 1929(penicillin), well over 2,000 different types of antibiotic substancehave been discovered, although the toxicity and side effects of many ofthem mean that only around 30 are used widely as chemotherapeutic drugs.The most effective antibiotics are frequently artificially producedderivatives of natural vegetable (from fungi and algae) or animalantibiotics; completely synthetic antibiotics are also available.Antibiotics act in very different ways: Some antibiotics positivelydestroy the microbes; others lead to the “quiet” death of the cellwhereas others are only growth inhibitors and leave the combating anddestruction of the inhibited, and hence weakened, microbes to thehealing immune system of the patient concerned, whether human, animal orplant.

In the middle of the last century, antibiotics were acclaimed as thegreat hope for fighting infectious diseases, whereas they are nowthreatening to quickly become a blunt instrument. The only hope ofsalvation is through targeted use with courses of treatment which arecompleted in full, and this requires rapid identification of theinfective pathogens and also the fast identification of their specificresistances to various types of antibiotics.

SUMMARY

The invention is based on the mass spectrometric measurement of themodification of the protein profile effected by antibiotics during abrief growth phase in a good nutrient medium under conditions which areotherwise ideal. It is preferable if liquid nutrient media are usedsince they enable the microbes to multiply quickly, at any rate fasterthan they would on the surface of a gelatinous medium. The effect of theantibiotics can generally be measured clearly after only around twohours. To give an example, bacteria divide every 20 to 30 minutes; intwo hours they can multiply by factors of between 16 and 64.

After the period allowed for the antibiotics to act, the microbes areseparated from the nutrient medium by filtration or preferably bycentrifuging, applied to a mass spectrometric sample support, sprinkledwith matrix solution and fed to the mass spectrometer when dry. The massspectra measured here can be compared with spectral libraries which alsocontain the mass spectra of resistant and non-resistant microbes afterantibiotics have acted on them. But it is often sufficient simply tocompare them with the mass spectra of microbes cultivated in the sameway but without being subjected to the effect of antibiotics.

If the antibiotics have a destructive effect on the microbes, then thedifference between resistant and non-resistant microbes can easily beseen in the mass spectrum (and can also be determined by computerprograms). The situation is different when the microbes simply die offwithout their proteins being destroyed or greatly changed (somethingwhich seldom occurs, however), or when the growth of the microbes issimply disrupted and they remain alive in the nutrient medium but do notmultiply further. In such cases, reactive substances can be added to thenutrient media containing antibiotics, said substances boosting andassisting the effect of the antibiotics. It is possible, for example, toadd enzymes which can attack and destroy microbes whose growth has beenaffected while unaffected microbes cannot be attacked by the enzymes.

Alternatively, substances can also be added to the nutrient media whichhighlight the difference between newly grown and growth-inhibitedmicrobes mass spectrometrically. It is possible, for example, to addisotope-marked nutrients which bring about a characteristic modificationof the mass profile of the proteins. If the growth of the microbes isinhibited and they absorb no nutrients, this is immediately identifiedmass spectrometrically because the modifications are absent.

Adding the same quantity of reference microbes of a similar type andusing a quantitative reference measurement can also serve to identifygrowth-inhibited or even dead microbes, but not those which are lyzed.In two hours, the difference between the number of non-growing andgrowing microbes has increased by at least a factor of 10 and so it ispossible to identify growth-inhibited microbes because there are fewerof them in the mass spectrum.

The method can be used on several portions of the nutrient medium whichare used in parallel and supplied with different types of antibiotic insuch a way that several types of resistance can be measured at the sametime, for example in microtitration plates which can also becentrifuged. Responsible hospitals generally only use between three anda maximum of five antibiotics on a regular basis, with some five furtherantibiotics on hand for cases of resistance, so that only theresistances to ten antibiotics have to measured on a regular basis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows two mass spectra with protein profiles of Escherichia coli:the mass spectrum at the top is from a non-resistant type whereas the E.coli in the mass spectrum at the bottom has a resistance to ampicillindue to a plasmid pUC19. The mass spectra contain predominantly proteinsignals of the same mass, with the usual lack of reproducibility of theintensities. They are identified as being identical by suitable computerprograms.

FIG. 2 shows the protein profiles of the non-resistant (top) andresistant (bottom) E. coli from FIG. 1 except that, in this case,ampicillin in a liquid nutrient medium has been allowed to act for twohours. The non-resistant type of E. coli (top) has been destroyed; allprotein signals of the mass spectra from FIG. 1 have disappeared. Theresistant type (bottom) produces a protein profile which is sufficientlysimilar to the two mass spectra in FIG. 1.

FIG. 3 shows a flowchart illustrating the steps in an illustrativeprocess for determining the resistance of microbes to a specificantibiotic in accordance with the principles of the invention.

DETAILED DESCRIPTION

While the invention has been shown and described with reference to anumber of embodiments thereof, it will be recognized by those skilled inthe art that various changes in form and detail may be made hereinwithout departing from the spirit and scope of the invention as definedby the appended claims.

As illustrated in FIG. 3, the invention provides a method of determiningthe resistance of microbes to a specific antibiotic. The illustrativemethod starts in step 300 and comprises the following steps:

(a) In step 302, a quantity of microbes under investigation is added toa nutrient medium which also contains a quantity of the antibiotic,

(b) Next, in step 304, the microbes are incubated at a predeterminedtemperature for a predetermined length of time, preferably not longerthan a few hours,

(c) In step 306, the microbes are then removed from the nutrient mediumby a suitable means, such as centrifuging,

(d) The microbes are then applied, in step 308, together with a matrixsolution to a mass spectrometric sample support and the matrix solutionis dried on the sample support to form a sample,

(e) In step 310, a mass spectrum of this sample is acquired, and

(f) Then, in step 312, the acquired mass spectrum is compared withreference mass spectra of these microbes. The process then finishes instep 314.

The invention is thus based on the mass spectrometric measurement of themodification of the mass spectrum of these microbes effected byantibiotics during a relatively brief growth phase in a good nutrientmedium under conditions which are otherwise as ideal as possible. Themass spectrum essentially represents the profile of the soluble proteinsin the interior of the microbes; the non-soluble membrane proteins ofthe microbes are generally not visible. It is also quite possible thatsome substances which are not proteins are represented in the massspectrum; for the sake of simplicity, however, the term “proteinprofiles” will be used below, said profiles being represented in themass spectra of the microbes.

The method generally begins with an identification of the microbes, asdescribed above: microbes are cultivated on gelatinous nutrient mediaand a portion of the microbes of a colony is used to identify themicrobes by means of their mass spectrum. This identification is notabsolutely necessary, but it can be helpful for the subsequentdetermination of the resistances according to this invention because itmay modify the measurement of the resistances depending on the type ofthe microbes. It can determine the temperature which must be maintainedfor optimum growth in the nutrient medium, for example.

Since the parallel identification of a large number of microbes byacquiring the many mass spectra takes around two hours, this time can beused to multiply the remaining microbes from the selected colonies byincubation. It is preferable to use liquid nutrient media since theyenable the microbes to multiply quickly. This type of reproduction isgenerally much quicker than cultivating the microbes on the surface of agelatinous nutrient medium. Bacteria in a good liquid nutrient medium atoptimum temperatures, for example, divide every 20 to 30 minutes, sothat in two hours, they can multiply by factors of between 16 and 64.The liquid nutrient media are commercially available.

Further portions of the colony or portions of the microbes of the colonywhich have been multiplied by incubation can then be used to determinethe resistances to different types of antibiotic by incubating them innutrient media to which the prescribed antibiotics have been added.Here, as well, it is preferable to use liquid nutrient media. Themicrobes of a colony, which have multiplied in the meantime by beingincubated in liquid nutrient media, can be easily distributed over adozen or so vessels by simple pipetting, said vessels containing theliquid nutrient media together with antibiotics. The effect of theantibiotics can generally be clearly measured after approximately twohours. It is particularly favorable if one of the vessels contains noantibiotic; it is then very easy to obtain a reference mass spectrum forthe purposes of comparing microbes which have the same history. This isparticularly favorable if a spectral library with correspondingreference mass spectra cannot be used, or if these microbes could not beidentified in the spectral library because it did not contain acorresponding reference mass spectrum.

Infectious microbes are incubated for about two hours at temperatures ofbetween 35° C. and 40° C. in the presence of the antibiotics. Thistemperature is the optimum one for most infectious microbes because theyare generally adapted to a life in mammals. Other types of microbes growbest at other temperatures, so it is favorable to identify the microbesbeforehand. The microbes are then separated from the nutrient medium,preferably by centrifuging, and are precipitated as sediment. Thisprocess of cultivation and removal can be carried out for severalantibiotics in parallel in the microvessels of microtitration plates,and the centrifuging can also be done in these microtitration plates.Alternatively, the microbes can also be separated off by filtration orother suitable separation methods.

As is the case with the identification, the separated microbes are thenapplied to a mass spectrometric sample support, sprinkled with matrixsolution and fed to the mass spectrometer when the matrix substance hasdried and crystallized out. Matrix solution can also be added directlyto the sediments after the supernatants have been removed, the matrixsolution being pipetted onto the mass spectrometric sample support withthe proteins taken up.

It is preferable if the mass spectra measured in the mass spectrometerare compared with spectra in spectral libraries which also contain themass spectra of resistant and non-resistant microbes after antibioticshave acted on them. These mass spectra can each contain furtherinformation such as threshold values for the protein signals which haveto be achieved. However, there is generally such a dramatic differencebetween the mass spectra of resistant and non-resistant microbes that itsuffices just to compare them with the mass spectra of microbescultivated in the same way but without being subjected to the effect ofantibiotics.

The antibiotics can act on the microbes in a variety of ways: They cancompletely destroy them (“lysis”), they can kill them off withoutdestroying the cell membrane, or they can simply inhibit their growth sothat they can practically no longer multiply. The microbes whose growthis inhibited are generally considerably weakened.

The destruction of the microbes is immediately visible in the massspectrum because the mass spectrum now bears no similarity whatsoever tothe microbe spectra of the living microbes. Such a case is shown inFIGS. 1 and 2 for E. coli under the effect of ampicillin. The proteinsof the microbes are lost because the centrifuging precipitates only themembrane sheaths in the main. Most antibiotics completely destroy themicrobes.

When the microbes are killed off leaving their cellular structureintact, and also to a certain extent when growth is inhibited,considerable changes to the internal metabolism occur. The proteases areno longer controlled and so the nucleoproteins of the ribosomes, whichare present in high concentrations, and other proteins present in highconcentrations, are immediately broken down. This means that the massspectra which are measured are very different but it is quite possiblethat they still have several of the protein signals which are found inhealthy, living microbes.

If growth is only weakly inhibited, many proteins in the microbes remainintact and can thus also be found in an unmodified state in the massspectra. Only a few proteins have recognizable modifications: enzymaticattacks (many antibiotics are enzymes), mainly by the above-describedendogenous proteases, change the mass of the proteins and so they appearin a different place in the mass spectrum. It is much more difficult toidentify the resistance in this case than it is with microbes which havebeen completely destroyed. The dead or growth-inhibited microbes nowgenerally have weakened membranes, however, so that other substanceswhich would normally not have a damaging effect on the microbes can nowpenetrate into the microbes and bring about characteristicmodifications, for example digestion of the proteins. One embodiment ofthe invention is therefore to add other attacking substances to thenutrient medium at the same time as the antibiotics, for exampledigestion enzymes such as proteases.

A lack of growth due to the effect of the antibiotics can also bedetermined in other ways. One option is to add substances to thenutrient media which make it possible to differentiate massspectrometrically between newly grown and growth-inhibited microbes. Itis possible, for example, to add isotope-marked nutrients which bringabout a characteristic modification of the mass profile of the proteinsin living and growing microbes. All amino acids in the nutrient mediacan be marked with the ¹⁵N isotope of nitrogen, for example. If thegrowth of the microbes is inhibited and they absorb no nutrients, thisis immediately identified mass spectrometrically because thecharacteristic mass increases brought about by ¹⁵N are absent. It isalso possible to undertake other types of derivatization of nutrientsfrom the nutrient media, however. Nutrients are known whose derivatesare taken up fully by microbes into the metabolism instead of theoriginal nutrients, thus forming products of different mass.

Moreover, a lack of growth can also be clarified by quantitative growthcomparisons. If it is not possible to differentiate between the proteinprofiles of inhibited and normal microbes, then the addition of the samequantity of reference microbes of a similar type and the use of aquantitative reference measurement can serve to identifygrowth-inhibited or even dead microbes, but not those which are lyzed.It is preferable if the reference microbes are resistant and that theygrow normally; if necessary, non-resistant microbes can also be used,however. The mass spectra of the two types of microbe are superimposedroughly 1:1 if both types of microbe are present in the samplepreparation in equal quantities. In two hours, the difference betweenthe number of non-growing and growing microbes has increased by at leasta factor of 10 and so it is possible to identify growth-inhibitedmicrobes by how many of them are present in the mass spectrum. It iseven possible to identify growth-inhibited microbes when their growth isnot completely inhibited but they merely grow significantly more slowlythan resistant strains. An advance identification can provideinformation as to whether such semi-resistant types of microbe may bepresent.

The method can be used on several portions of the nutrient medium whichare used in parallel and supplied with different types of antibiotic insuch a way that several types of resistance can be measured at the sametime, for example in microtitration plates which can also becentrifuged. In this case it is advisable to allow the microbes to growin one of the microvessels without antibiotics being added in order toobtain reference mass spectra of these microbes.

Responsible hospitals generally only use between three and a maximum offive antibiotics on a regular basis, with some five further antibioticson hand for cases of resistance, so that only the resistances to aboutten antibiotics have to measured on a regular basis.

With knowledge of the invention, the methods described here can bemodified by those skilled in the art in a wide variety of ways. Some ofthese modifications have already been described above; there arecertainly further methods which, on the fundamental basis of a briefcultivation, can generate the desired informative mass spectra of themicrobes with information about their resistances.

What is claimed is:
 1. A method for determining the resistance to aspecific antibiotic of microbes having a known mass spectrum whencultivated in a first growth medium without an antibiotic, comprising:cultivating a sample of the microbes in a growth medium substantiallythe same as the first growth medium that additionally contains theantibiotic and a nutrient which, if absorbed by the microbes, producescharacteristic modifications in the known mass spectrum; acquiring amass spectrum of the cultivated sample; and determining the microbes tobe resistant to the antibiotic if the characteristic modifications arepresent in the mass spectrum.
 2. The method of claim 1, wherein thegrowth medium is a liquid growth medium.
 3. The method of claim 2,wherein the liquid growth medium is contained in a microvessel of amicrotitration plate.
 4. The method of claim 2, further comprisingremoving the cultivated microbes from the growth medium by filtration orcentrifuging.
 5. The method of claim 1 wherein the nutrient is asubstance marked with one or more isotopes.
 6. The method of claim 5wherein said substance marked with one or more isotopes comprises aminoacids.
 7. The method of claim 6 wherein the amino acids are marked withthe ¹⁵N isotope of nitrogen.
 8. The method of claim 5 wherein thecharacteristic modifications comprise mass increases.
 9. A method fordetermining the resistance of microbes to a specific antibiotic,comprising: (a) cultivating a first quantity of the microbes in a growthmedium; (b) cultivating a second quantity of the microbes using the samegrowth medium and cultivation conditions used in step (a) except thatthe growth medium contains the antibiotic and marked nutrients whoseintake by the microbes results in a detectable difference in a massspectrum of the microbes; (c) preparing a first sample from the microbescultivated in step (a) using whole microbes or proteins extracted fromthe microbes; (d) preparing a second sample from the microbes cultivatedin step (b) using whole microbes or proteins extracted from themicrobes; (e) measuring a first mass spectrum of the first sample thatis indicative of the soluble proteins of the microbes cultivated in step(a); (f) measuring a second mass spectrum of the second sample that isindicative of the soluble proteins of the microbes cultivated in step(b); (g) comparing the first and second mass spectra; and (h)determining the resistance of the microbes based on the comparison ofstep (g), wherein an absence of said detectable difference in the secondmass spectrum indicates a growth-inhibition of the microbes.
 10. Themethod of claim 9, wherein the growth medium is a liquid growth medium.11. The method of claim 10, wherein the liquid growth medium iscontained in a microvessel of a microtitration plate.
 12. The method ofclaim 10, further comprising removing the cultivated microbes from thegrowth medium by filtration or centrifuging.
 13. The method of claim 9wherein the marked nutrients are substances marked with isotopes. 14.The method of claim 13 wherein all amino acids in the growth medium aremarked with isotopes.
 15. The method of claim 14 wherein the amino acidsare marked with the ¹⁵N isotope of nitrogen.
 16. The method of claim 13wherein said detectable difference comprises a mass increase.